Designer rhamnolipids by reduction of congener diversity: production and characterization

Abstract

Background: Rhamnolipids are biosurfactants featuring surface-active properties that render them suitable for a broad range of industrial applications. These properties include their emulsification and foaming capacity, critical micelle concentration, and ability to lower surface tension. Further, aspects like biocompatibility and environmental friendliness are becoming increasingly important. Rhamnolipids are mainly produced by pathogenic bacteria like Pseudomonas aeruginosa. We previously designed and constructed a recombinant Pseudomonas putida KT2440, which synthesizes rhamnolipids by decoupling production from host-intrinsic regulations and cell growth.

Results: Here, the molecular structure of the rhamnolipids, i.e., different congeners produced by engineered P. putida are reported. Natural rhamnolipid producers can synthesize mono- and di-rhamnolipids, containing one or two rhamnose molecules, respectively. Of each type of rhamnolipid four main congeners are produced, deviating in the chain lengths of the β-hydroxy-fatty acids. The resulting eight main rhamnolipid congeners with variable numbers of hydrophobic/hydrophilic residues and their mixtures feature different physico-chemical properties that might lead to diverse applications. We engineered a microbial cell factory to specifically produce three different biosurfactant mixtures: a mixture of di- and mono-rhamnolipids, mono-rhamnolipids only, and hydroxyalkanoyloxy alkanoates, the precursors of rhamnolipid synthesis, consisting only of β-hydroxy-fatty acids. To support the possibility of second generation biosurfactant production with our engineered microbial cell factory, we demonstrate rhamnolipid production from sustainable carbon sources, including glycerol and xylose. A simple purification procedure resulted in biosurfactants with purities of up to 90%. Finally, through determination of properties specific for surface active compounds, we were able to show that the different mixtures indeed feature different physico-chemical characteristics.

Conclusions: The approach demonstrated here is a first step towards the production of designer biosurfactants, tailor-made for specific applications by purposely adjusting the congener composition of the mixtures. Not only were we able to genetically engineer our cell factory to produce specific biosurfactant mixtures, but we also showed that the products are suited for different applications. These designer biosurfactants can be produced as part of a biorefinery from second generation carbon sources such as xylose.

Keywords: Bioeconomy; Designer biosurfactants; Hydroxyalkanoyloxy alkanoate; Non-pathogenic Pseudomonas; Rhamnolipid.

Fig. 1

a Structure of rhamnolipids. The upper part of the molecule is formed by the hydrophobic moiety, the hydroxyalkanoyloxy alkanoate (HAA). The chain lengths of the β-hydroxy-fatty acids in this dimer can vary. One or two rhamnose molecules are bound by a glycosidic bond. The sugar molecules are the hydrophilic moiety of the molecule. Molecules with one rhamnose are called mono-rhamnolipids, while the here depicted molecule with two rhamnoses is a di-rhamnolipid. b Biosynthesis pathways of rhamnolipids. Based on glucose two pathways are required for rhamnolipid synthesis. In the lower part, activated β-hydroxy-fatty acids are formed via fatty acid de novo synthesis, which are fused by the enzyme RhlA. In the upper part, activated rhamnose is synthesized and subsequently coupled to the β-hydroxy-fatty acid dimer by the rhamnosyltransferase I (RhlB). The rhamnosyltransferase II (RhlC) finally adds another sugar molecule to yield a di-rhamnolipid. Enzyme names are printed in grey

Fig. 2

a Surfactant congeners produced by the different microbial cell factories. The striped columns represent the congener distribution for the HAAs produced by P. taiwanensis VLB120, the grey columns show the congener for the mono-rhamnolipids, and the black columns depict di-rhamnolipid congeners. b HAA production with recombinant P. putida KT2440 pSB01. The courses of CDW and HAA generation are shown over the fermentation time. The black filled rectangles and the dashed line depict HAA titers, while the gray triangles represent the biomass concentrations. The error bars represent deviation from the mean of two replicates

Fig. 3

Mono-rhamnolipid production using alternative carbon sources. CDW and rhamnolipid titers are presented in g/L (striped columns and grey columns, respectively). The error bars represent the deviation from the mean of two biological replicates

Fig. 4

Purification of HAAs using an adsorption/desorption procedure. The composition of the different fractions collected is shown. Each fraction corresponds to 10 min of elution. The black curve with the squares displays the total HAA concentration in g/L. The black dashed line with the circles shows the pyoverdine content in arbitrary units, while the black dotted line with triangles represents other impurities measured by absorption at 400 nm in arbitrary units. The gray line with squares displays the purity. The grey area in the background shows the composition of the elution solution with stepwise increasing ethanol concentrations. The stacked bars display the congener’s composition of the HAAs. Black bars: C₁₀-C₈, white bars: C₁₀-C₁₀, dark grey bars: C₁₀-C_12:1, light grey bars: C₁₀-C₁₂ (all in g/L)

Fig. 5

Purification of mono-rhamnolipids using an adsorption/desorption procedure. The composition of the different fractions collected is shown. Each fraction corresponds to 10 min of elution. The black curve with the squares displays the total rhamnolipid concentration in g/L. The black dashed line with the circles shows the pyoverdine content in arbitrary units, while the black dotted line with triangles represents other impurities measured by absorption at 400 nm in arbitrary units. The gray line with squares displays the purity. The grey area in the background shows the composition of the elution solution with stepwise increasing ethanol concentrations. The stacked bars display the congener’s composition of the rhamnolipids. Black bars: Rha-C₈-C₁₀, white bars: Rha-C₁₀-C₁₀, dark grey bars: Rha-C₁₀-C_12:1, light grey bars: Rha-C₁₀-C₁₂ (all in g/L)

Fig. 6

Purification of di-rhamnolipids using an adsorption/desorption procedure. a The composition of the different fractions collected is shown. Each fraction corresponds to 10 min of elution. The black curve with the squares displays the total rhamnolipid (mono and di) concentration in g/L. The black dashed line with the circles shows the pyoverdine content in arbitrary units, while the black dotted line with triangles represents other impurities measured by absorption at 400 nm in arbitrary units. The gray line with squares displays the purity. The grey area in the background shows the composition of the elution solution with stepwise increasing ethanol concentrations. The stacked bars display the congener’s composition of the di-rhamnolipids. Black bars: Rha-Rha-C₈-C₁₀, white bars: Rha-Rha-C₁₀-C₁₀, dark grey bars: Rha-Rha-C₁₀-C_12:1, light grey bars: Rha-Rha-C₁₀-C₁₂ (all in g/L). b Mono-rhamnolipids contained in the fractions. The black line depicts the total mono-rhamnolipid concentration, while the stacked bars represent the amount of mono-rhamnolipid congeners. Black bars: Rha-C₈-C₁₀, white bars: Rha-C₁₀-C₁₀, dark grey bars: Rha-C₁₀-C_12:1, light grey bars: Rha-C₁₀-C₁₂ (all in g/L)

Fig. 7

Biosurfactant properties. a Foaming capabilities I. Course of the foam formation over time. HAAs are represented by the light grey dashed line with circles, mono-rhamnolipids by the dotted grey line with triangles, di-rhamnolipids by the dark grey line with diamonds, SDS is depicted by black line with squares and serves as a reference. b Foaming capabilities II. Amount of added antifoam during the testing of the antifoam effectiveness. The error bars represent the deviation of the mean of three experiments. c Emulsification capability. Volumetric percentage of the different phases foam, oil, emulsion, and aqueous phase of the initial emulsion. d Emulsion stabilization. Volumetric change over time of the different phases foam, oil, emulsion, and aqueous phase

Fig. 8

Congener composition of the biosurfactant mixtures produced by the three different production cell factories. The first bar represents the composition in the HAA producing cell factory carrying only rhlA. The second cell factory also carries rhlB and is thus capable of producing mono-rhamnolipids, while the third cell factory produces di- and mono-rhamnolipids, mediated by the second rhamnosyltransferase rhlC. White columns depict the share of the congener being composed of C₁₀-C₈ β-hydroxy-fatty acids. The striped box represents the share of C₁₀-C₁₀ hydrophobic moieties, while the grey and black fields stand for C₁₀-C_12:1 and C₁₀-C₁₂ hydrophobic moieties of the biosurfactant molecule, respectively. The error bars represent the deviation from the mean and are based on the values of ten time points from two biological replicates